Enzymatic deacylation of benzyl- and phenoxymethylpenicillin tetrazoles

ABSTRACT

Deacylation of benzyl- and phenoxymethylpenicillin tetrazoles to produce 6-amino-2,2-dimethyl-3-(tetrazol-5-yl)penam by the use of selected deacylases. The process is useful in the purification of said penam which is a valuable synthetic intermediate. It has been found that those enzymes which will deacylate benzyl- and phenoxymethylpenicillin will generally deacylate the corresponding tetrazoles.

United States Patent [191 Hamsher [4 1 Sept. 16, 1975 ENZYMATIC DEACYLATION OF BENZYL- AND PHENOXYMETHYLPENICILLIN TETRAZOLES [75] Inventor: James J. Hamsher, Gales Ferry,

Conn.

[73] Assignee: Pfizer Inc., New York, NY.

[22] Filed: Dec. 4, 1974 [21] Appl. No.: 529,480

[52] US. Cl. 195/36 P; 260/2391; 260/3067 C [51] Int. Cl. C12D 13/02 [58] Field of Search.. 195/36 P; 260/2391, 306.7 C

[56] References Cited UNITED STATES PATENTS 3,736,230 5/1973 Delin 195/36 P Primary Examiner'-Alvin E. Tanenholtz Attorney, Agent, or Firm-Connolly and Hutz [5 7] ABSTRACT 33 Claims, No Drawings ENZYMATIC DEACYLATION OF BENZYL- AND PHENOXYMETHYLPENICILLIN TETRAZOLES BACKGROUND OF THE INVENTION 1. Field Of The Invention It is known that certain compounds of the structure:

H CH3 R -N N O \N NJ R'/ and H CH3 R -N- and their salts wherein R is selected from the group consisting of the acyl moieties of phenylacetic and phenoxyacetic acid;

and R' and R;; are each selected from the group consisting of hydrogen, alkanoyloxymethyl having from three to eight carbon atoms, l-(alkanoyloxy)ethyl having from four to nine carbon atoms and phthalidyl;

wherein R is selected from the group consisting of R' trialkylsilyl having up to four carbon atoms in each alkyl group and a tetrazolylpenam nitrogen protecting group which is defined hereinafter;

R is selected from the group consisting of R';, and trialkylsilyl having up to four carbon atoms in each alkyl group;

and R is selected from the group consisting of R hydrogen, and an amine protecting group which is defined hereinafter;

are valuable as intermediates in the synthesis of said antibacterial agents.

These compounds are disclosed in the copending, commonly assigned application Ser. No. 491,510, filed July 24, 1974, the disclosure of which is incorporated here by reference.

2. The Prior Art Benzyland phenoxymethylpenicillin are produced in fermentation processes by a wide variety of organ- -isms. These are the most common of the naturallyoccurring penicillins. Semisynthetic penicillins differ from the naturally-occurring penicillins usually only in the nature of the N-acyl group. These are prepared by deacylating benzylpenicillin or phenoxymethylpenicillin to form 6-amino-2,2-dimethylpenicillanic acid and phenylacetic or phenoxyacetic acid respectively and then reacylating said penicillanic acid with the desired acyl moiety; both of these processes have been carried out by microbial methods. Many valuable semisynthetic penicillins have been produced in this manner.

The present invention is directed to the enzymatic deacylation of penicillin tetrazoles. Since penicillin tetrazoles were unknown in the art prior to Ser. No. 407,097, there is obviously no prior art on the deacylation of penicillin tetrazoles. The enzymatic deacylation of benzyl and phenoxymethylpenicillin has been effected in the past. However, because enzymes are known to be highly specific, one skilled in the art would in no way expect that the same enzymes effective on these substrates would be effective with the corresponding tetrazoles. The deacylation of penicillins has been reviewed in Cephalosporins and Penicillins, ed. E. H. Flynn, Academic Press (New York, 1972).

SUMMARY OF THE INVENTION A process is disclosed for the enzymatic deacylation of penicillin tetrazoles of the structure wherein R R and R are as previously defined. The process is also applicable to the salts of these compounds. The process comprises dispersing said penicillin tetrazoles in water at a concentration of at least 0.1% by weight, maintaining the pH of the reaction medium between about 5 and 9 and the temperature between 5 and 50C. while contacting said penicillin tetrazole with a deacylase selected as described below until the reaction to form the product with the general structural formula I or II wherein R, is hydrogen is substantially complete. In those compounds wherein R or R are subject to solvolysis in water or mild alkaline solutions'such as those wherein R or R is trialkylsilyl and certain of the tetrazolylpenam nitrogen protecting groups, a product will be obtained wherein R or R is hydrogen. In the preferred embodiment, the penicillin tetrazole concentration is between about 0.1 and 20.5% by weight, the temperature is maintained between 25 and 45C. and the pH between 7.0 and 8.8. The deacylase is introduced into the reaction medium by a member of the group comprising bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, enzymes from said bacteria or fungi and said enzymes immobilized on a matrix. Microorganisms of the genera Proteus," Escherichia, kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus which successfully deacylate penicillin tetrazoles are disclosed. Said deacylases are also acylases for benzylpenicillin and the salts thereof when R, is the acyl moiety of phenylacetic acid and are acylases for phenoxymethylpenicillin and the salts thereof when R, is the acyl moiety of phenoxyacetic acid. Further, it has been surprisingly found that said deacylases are actually more active on the tetrazolylpenam substrate than they are on the corresponding penicillin substrate.

DETAILED DESCRIPTION OF THE INVENTION lnaccordance with the present invention, it has been found that all of those microorganisms which deacylate benzyland phenoxymethylpenicillin generally also deacylate the corresponding tetrazole compounds of the structure and the salts thereof, wherein R,, R and R are as previously defined.

In addition to the microorganisms'which include both bacteria and yeasts, whole cells of said microorganisms immobilized on a matrix, extracts and enzymes derived from said microorganisms and said enzymes immobilized on a matrix also effect the desired deacylation. The product of said enzymatic deacylation is a compound of the general structural formula III or IV wherein R is hydrogen. It should be noted that in those cases wherein R or R are subject to solvolysis in aqueuus solution, such as those wherein R or R are trialkylsilyl or certain tetrazolylpenam nitrogen protecting groups noted hereinafter, R or R may be displaced by hydrogen to yield a product wherein not only R, but also R or R are hydrogen. This solvolytic displacement is an artifact of the solvent and totally incidental to the enzymatic deacylation which is of interest in the instant invention. In addition, whenever R or R is hydrogen, compounds of the general structural formulae V and VI exist in a tautomeric equilibrium; this is not the case for any other R or R substituent.

Because of the well-known high specificity of enzymes, the process of the instant invention is surprising indeed. It would be unforseen by one skilled in the art of enzymology that all of those enzymes which deacylate benzyland phenoxymethylpenicillin will also deacylate the respective tetrazole and substituted tetrazole analogues. One might expect that some insignificant amount of deacylation might occur in the tetrazole case but one would hardly expect these microorganisms and the enzymes derived therefrom to be more active in the tetrazole case. It is believed that enzyme systems evolve in response to or are selected by environmental pressures. This theory does not imply that natural pressures create mutations but rather that they select those variants which cope best with these environmental pressures. Since benzyland phenoxymethylpenicillin are naturally occurring substances toxic to many microorganisms and since the biocidal tetrazole analogues are laboratory artifacts, one would be fully justified in expecting in view of the theory of natural selection that any enzyme system capable of detoxifying penicillins would be more effective on the naturally occurring substrate than on the tetrazole analogue. Furthermore, steric factors are often of prime importance in predicting the activity of an enzyme with various substrates. In simplest terms, the substrate must be able to fit into the active site on the enzyme if activity is to be observed. The acid moiety on the natural penicillins is appreciably smaller in size than the tetrazole or protected tetrazole moiety. Therefore, a consideration of steric factors would lead one to the same conclusions as a consideration of evolutionary factors: naturally occurring penicillin deacylases should exhibit either no activity or significantly diminished activity with penicillin tetrazoles rather than natural penicillins as a substrate. In fact, it has been surprisingly found not only that all the deacylases are active on a tetrazole substrate but also that activities equal to and, in some instances, threefold greater are observed with the tetrazole substrate.

The term tetrazolylpenam nitrogen protecting group" is intended to connote all groups known, or obvious, to one skilled in the art, which can be used (a) to permit the synthesis of the compounds of formula III, wherein R is an amino protecting group and R is the said tetrazolylpenam nitrogen protecting group, by the process starting with 6-(protected amino)penicillanic acid described hereinafter; and (b) can be removed from a compound of formula I, wherein R, is an acyl group and R is the said tetrazolylpenam nitrogen protecting group, or from a compound of formula III, wherein R is selected from the group consisting of hydrogen and an amino protecting group, and R is the said tetrazolylpenam nitrogen protecting group, using a method wherein the penam ring system remains substantially intact. The tetrazolylpenam nitrogen protecting group is required in order to protect the nitrogen atom which ultimately becomes N-l of the tetrazole ring in the said compounds of formulae 1 through VI, during the conversion of a 6-(protected amino)penicillanic acid into a compound of formula III. It is the ability of the tetrazolylpenam nitrogen protecting group to perform a specific function rather than its precise chemical structure, which is important; and the novelty of the antibacterial agents of the invention does not depend upon the structure of the protecting group. Selection and identification of appropriate protecting groups can be made readily and easily by one skilled in the art.

An example of a typical tetrazolylpenam 'nitrogen protecting group is -CH2CH /Y wherein Y is an electron-withdrawing group, and Y is either hydrogen or a further electron-withdrawing group, which can be the same as or different from Y. The function of the electron-withdrawing group is to render a hydrogen atom, on the carbon atom to which Y and Y are attached, sufficiently acidic that the group is removable in a retrograde Michael reaction. Such a reaction is well-known in the art. For example consult House, Modern Synthetic Reactions, W. A. Benjamin, Inc. New York/Amsterdam, 1965, page 207. Typical electron-withdrawing groups are cyano, alkoxycarbonyl having from two to seven carbon atoms, phenoxycarbonyl, alkylsulfonyl having from one to six carbon atoms, phenylsulfonyl and SO -NR R wherein R and R are each selected from the group consisting of hydrogen, alkyl having from one to four carbon atoms, phenyl and benzyl. A particularly convenient configuration for this protecting group is that wherein Y is hydrogen; and preferred values for Y are alkoxycarbonyl having from two to seven carbon atoms and phenylsulfonyl.

A further tetrazolylpenam nitrogen protecting group which can be used is a grouping of formula (C- =O)-OR Such a grouping can be removed by mild hydrolysis, such as mild alkaline hydrolysis, or treatment with a nucleophile, such as an amine, or a thiol or thiolate anion. Although a wide variety of groups can serve as R particularly convenient values are alkyl having from one to six carbon atoms, benzyl, phenyl" and substituted phenyl, for example, phenyl substituted by up to two moieties each selected from the group consisting of nitro, fluoro, chloro, bromo, alkyl having from one to four carbon atoms and alkoxy having from one to four carbon atoms.

A still further tetrazolylpenam nitrogen protecting group which can be used is a grouping of formula SO R,,. Such a group is also removed by hydrolysis, or by treatment with a nucleophilic agent, as indicated for the group (C=O)-OR and convenient values for R are also alkyl having from one to six carbon atoms, benzyl, phenyl and substituted phenyl, for ex ample, phenyl substituted by up to two moieties each selected from the group consisting of nitro, fluoro, chloro, bromo, alkyl having from one to four carbon atoms and alkoxy having from one to four carbon atoms. These two groups SO R,, and (C- =O)O-R are among those which wil be removed by solvolysis during deacylation.

A yet further tetrazolylpenam nitrogen protecting group which can be used is Particularly convenient configurations for this protecting group are 11 and I R13 I wherein R and R are each selected from the group consisting of hydrogen, hydroxy, nitro, fluoro, chloro, bromo, iodo, alkyl having from one to six carbon atoms, alkoxy having from one tosix carbon atoms, al kanoyloxy having from two to seven carbon atoms, formyloxy, alkoxymethoxy having from two to seven carbon atoms, phenyl and benzyloxy;

R is selected from the group consisting of hydrogen, alkyl having from one to four carbon atoms and phenyl;

R and R are each selected from the group consisting of hydrogen and methyl;

and X is selected from the group consisting of oxygen and sulfur.

As will be recognized by one skilled in the art, other groups which will also stabilize the carbonium ion (W- CH-W can replace those cited above for W and W.

Still another tetrazolylpenam nitrogen protecting group which can be used is phenacyl or substituted phenacyl. Such a group is removed by reaction with a nucleophilic reagent, such as thiophenoxide. Typical phenacyl groups which can be used are those of formula wherein R is selected from the group consisting of by drogen, nitro, fluoro, chloro, bromo and phenyl.

Amine protecting groups referred to above are all those known, or obvious, in the art, particularly the art of peptide synthesis. In general, their function is to prevent unwanted substitution of the amino group and the opening of the B'lactam ring. It should be possible to remove them under rather mild conditions when the reaction: scheme is complete but they should be stable under all other conditions encountered throughout the reaction scheme. Typical examples are the 2,2,2- trihaloethoxy carbonyls and substituted and unsubstituted triphenylmethyl group.

A characteristic feature of compounds of formulas I, through VI wherein the tetrazole ring is hydrogen substituted is their ability to form salts. By virture of the acidic nature of a S-monosubstituted tetrazole, said compounds have the ability to form salts with basic agents, and the salts, referred to generically as tetrazolate salts, are to be considered within the scope of this invention. The salts can be prepared by standard techniques, such as contacting the acidic and basic components, usually in a 1:1 molar ratio, in an aqueous, non-aqueous or partially aqueous medium, as appropriate. They are then recovered by filtration, by precipitation with non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate. Basic agents which are suitably employed in salt formation belong to both the organic and inorganic types, and they include ammonia, organic amines, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides. Representative examples of such bases are primary amines, such as npropylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine and octylamine; secondary amines, such as diethylamine, N-methylaniline, morpholine, pyrrolidine and piperidien; tertiary amines, such as triethylamine, N,N-dimethylaniline, N- ethylpiperidien, N-methylmorpholine and 1,5- diazabicyclo[4.3.0]non-5-ene; hydroxides, such as sodium hydroxide, potassium hydroxide, ammonium hy droxide and barium hydroxide; alkoxides, such as sodium methoxide; carbonates, such as potassium carbonate and sodium carbonate; and bicarbonates, such as sodium bicarbonate and potassium bicarbonate.

When therapeutic use in mammals is being contemplated for a salt of said compounds, it is of course essential to use a pharmaceutically-acceptable salt. However, other salts are useful for a variety of other purpopses; such as, for example, isolating and purifying individual compounds, changing the solubility characteristics of an individual compound, and for interconverting pharmaceutically-acceptable salts with their nonsalt counterparts.

The process of the present invention finds its utility in the preparation of substantially pure 6-amino-2,2- dimethyl-3(5-tetrazolyl)penam. Semi-synthetic penicillins are currently prepared by the chemical acylation of 6-amino2,2-dimethyl-3-(carboxylic acid)penam. By employing the same acylation process, the tetrazole analogues of said semi-synthetic penicillins may be prepared using the product of the process of the instant invention as the substrate.

The preparation of substrates of Formulas III and IV are described in detail in Ser. No. 491,5 l0. In one synthetic scheme, o-aminopenicillanic acid is first contacted with the chloride of one of the above-mentioned amine protecting group in a reaction-inert anhydrous solvent in the presence of a suitable base such as triethylamine at from about 0 to 20C. for at least about minutes. Reaction-inert solvents are those which are without substantially adverse effect on reactants and products under the conditions employed. A preferred amine protecting group is triphenylmethyl. The protected aminopenicillanic acid is then activated, usually by conversion to a mixed anhydride, and contacted with an amine of the formula RQNHQ at about 0C. until the reaction to form the desired N-substituted amide is substantially complete. It is not necessary to isolate the protected aminopenicillanic acid before contacting it with the amine but rather contact can be effected directly in the initial reaction mixture. Upon completion of the reaction, the crude amide is isolated by in vacuo evaporation. The crude amide is then dissolved in pyridine and cooled to about -5C. Thionyl chloride is added dropwise and the reaction mixture allowed to warm to room temperature. The mixture is allowed to stand until the reaction to form the imino chloride is substantially complete. The crude product is isolated by in vacuo evaporation, redissolved in a reaction-inert solvent and cooled to about 5. At least an equivalent amount of a suitable azide as trimethylsilyl azide is then added with stirring and the reaction-mixture allowed to warm to room temperature. Stirring is continued until the reaction to form the protected tetrazole is substantially complete. Excess azide is then destroyed by the addition of a suitable aqueous base. The waterimmiscible layer containing the product is then separated, extracted with brine, treated with a desiccant and concentrated to dryness to afford the crude product. The product may be purified by liquid chromatography. The product may be converted to one of Formula III wherein R is hydrogen by treating it with p-toluenesulfonic acid in a reaction-inert solvent such as acetone. A product of pharmaceutical grade purity may be prepared by the acylation-deacylation cycle described below.

Compounds of Formula III wherein R is hydrogen may be prepared from those of Formula III wherein R is hydrogen and R is not, by treating them with trifluoroacetic acid at about 40C. for at least 30 minutes. This reaction yields 6-amino-2,2-dimethyl-3-(5- tetrazolyl)penam. The contaminants are probably products of the polymerization of the removed tetrazole nitrogen protecting group. Previously, purification required a series of extractions followed by lyophilization and another series of extractions. Finally, the product must be recrystallized. It has now been found that a pharmaceutical grade product may be prepared by the acyIation-deacylation cycle described below.

Compounds of Formula IV wherein R is other than hydrogen may be prepared by contacting a 6-protected amino-2,2-dimethyl-3-(S-tetrazolyl)penam with a compound of the formula R Cl in a reaction-inert solvent usually in the presence of a base at about room temperature until the reaction is substantially complete. N,N-dimethylformamide is suitable as a solvent and triethylamine as a base. This reaction affords a mixture of isomers of Formulas III and IV which may be separated by thin layer chromatography.

In the purification process, crude 6-amino-2,2- dimethyl-3-(5-tetra2olyl)penams with or without substituents on the tetrazole ring are first acylated with phenylacetyl chloride or phenoxyacetyl chloride. The crude material is first suspended in water and brought into solution by the slow addition of aqueous base. The pH is then lowered to about 7 with a mineral acid and the suspension clarified by suction filtration. Approximately a 10 percent molar excess of phenylacetyl or phenoxyacetyl chloride is then added with stirring while maintaining the pH between about 6 and 7 with dilute aqueous base. The mixture is stirred for about four hours at room temperature. The mixture is then cooled to about 10C., its pH adjusted to about 2 with hydrochloric acid and it is then extracted several times with chloroform. The combined organic extracts are then poured into about a 6:1. hexane/ether mixture. The white precipitate which forms is then filtered, washed with hexane and dried to afford substantially pure benzylor phenoxymethylpenicillin tetrazole.

In the deacylation step of the process, the benzylor phenoxymethylpenicillin tetrazole is dispersed in water at concentration of at least about 0.1% and less than about 20% by weight. The pH is then adjusted with acid or base so that it is in the range of about 5 to 9 and preferably in the range of about 7.0 to 8.8 by the addition of a suitable acid or base such as hydrochloric acid or sodium hydroxide. The deacylase activity is introduced as whole cells, immobilized whole cells, cellular extracts, enzyme concentrates, substantially pure enzymes or immobilized enzymes. These modes of introduction along with others are'well known to those skilled in the art or zymurgy. In the use of whole cells or immobilized whole cells, the weight ratio of cells to said tetrazoles should be in the range of about 0.2 to 10. When using any other sources of said deacylase activity, the amount introduced should exhibit about 10 to 100 units of activity against benzylpenicillin for each gram of said tetrazole' present in the reaction mixture. Said mixture is then incubated aerobically at a temperature of about to 50C. and preferably between about 25 and 45C. while maintaining the pH in the range of about to 9 and preferably about 7.0 to 8.8 by the addition of a suitable acid or base until the reaction to form the 6-amino-2,2-dimethyl-3-( 5- tetrazolyl)penam is substantially complete.

The progress of the reaction may be monitored by employing one of the thin layer chromatography systems listed below along with appropriate blanks. Semiquantitative estimates of the yield of 6-aminopenicillin tetrazole can also be made from these chromatograms.

The product is isolated by first acidifying the reaction mixture to a pH of about 2 with a suitable acid such as hydrochloric acid and extracting the acidified solution several times with a suitable water-immiscible organic solvent such as ethyl acetate. The pH of the aqueous layer is then raised about 4.5 with a suitable base such as sodium hydroxide and concentrated in vacuo to yield said 6-aminopenicillin tetrazole product. Depending on the identity of the R moiety removed, phenylacetic or phenoxyacetic acid may be isolated from the organic layer by evaporation in vacuo after drying over a suitable desiccant.

The microorganisms of the instant invention are kept on agar slants. To culture whole cells, a water extract from the slant is first incubated in an inoculum medium for about 24 hours and a portion of the inoculum is then transferred to a fermentation medium which is then aerobically incubated for approximately 24 hours. Cells may be harvested as needed by centrifugation. The deacylase activity of these organisms may be increased by methods well-known to those skilled in the art of zymurgy such as culturing at about 25C. rather than 37C. and adding phenylacetic or phenoxyacetic acid to the culture medium. Said activity may be extracted from cells with solutions such as 0.2 M sodium chloride or sodium citrate. The enzyme may then be precipitated by a variety of reagents such as ammonium sulfate, calcium nitrate together with a quaternary ammonium salt or acetone. Said enzyme may then be purified by dialysis followed by lyophilization or solidliquid chromatography. The preparation of immobilized enzymes has been fully discussed by O. Zaborsky in Immobilized Enzymes, CRC Press (Cleveland, 1 973 Species of the following genera were found to exhibit deacylase activity for benzyland phenoxymethylpenicillin tetrazoles and their carbamoyl analogues: Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus. The organisms listed below were found to contain said deacylase: Proteus rettgeri, ATCC 9918; Proteus rettgeri, ATCC 31052; Escherichia coli, ATCC 9637; Escherichia coli, ATCC 31030; Kluyvera citrophilia, ATCC 21 285 Acetobacter cerinus, [F0 3268; Aerobacter Aerogenes, ATCC 31027; Arthrobacler tumescensyATCC 6947; Bacillus subtilis, ATCC 31028; Bacillus species, ATCC 31029; Cryptococcus albidus, ATCC 10666. These organisms are on deposit at either the American Type Culture Collection of Rockville, Maryland or The Institute for Fermantation, Osaka and were assigned the registry numbers shown above. ATCC 31028 was originally identified and registered as B. globigii. Later biochemical tests indicate that it is actually B. subtilis. There is reason to believe that this culture may be a subtransfer of ATCC 9372 but it has not been possible to prove or disprove this. ATCC 31029 was originally identified and registered as B. mesentericus. Tests later indicated that it actually is an unidentifiable species or mixture of species of the genus Bacillus. All of these organisms, except Cryptacoccus albz'dus which is a yeast or fungus, are bacteria. As with benzyland phenoxymethylpenicillin, deacylases of bacterial origin appear to hydrolyze benzylpenicillin tetrazole more rapidly than phenoxymethylpenicillin tetrazole while the converse is true for those of fungal origin. 1

EXAMPLE I 6-(Triphenylmethylamino)-2,2-dimethyl-3-( l-[4- methoxybenzyl] tetraz-ol -5-yl)penam.

6-(Triphenylmethylamino)-2,2-dimethyl-3-(N-[4- methoxybenzyl1carbamoyl)penam To a stirred slurry of2 l 6 g. of 6-aminopenicillanic acid in 1,500 ml. of anhydrous chloroform is added 278 ml. of triethylamine, and the mixture is then stirred at ambient temperature until a clear solution is obtained. This requires about 15 minutes. The solution is cooled to about 0C., and then 306 g. of triphenylmethyl chloride is added. The stirring is continued at about 0C. for 30 minutes, and then at ambient temperature for a further 24 hours. The mixture is cooled to about 0C. again, and 14 ml. of triethylamine, followed by ml. of ethyl chloroformate, is added. During this process the temperature rises to about 15C., and a precipitate forms. To facilitate stirring a further 200 ml. of chloroform is added. The stirring is continued for 30 minutes. Then, at about 0C., 50 ml. of 4-methoxybenzylamine (available from the Aldrich Chemical Company, Inc.) is injected into the reaction medium, below the: surface of the solvent. At 10 minute intervals, three further aliquots of 4- methoxybenzylamine (35 ml., 25 ml. and 21 ml.) are injected in the reaction in similar fashion. The total volume of 4-methoxybenzylamine added is 131 ml. The cooling bath is then removed, and the reaction is stirred for a further 1 hour. The chloroform solution is washed successively with five 2,000-ml. portions of water and one 2,000-ml. portion of saturated brine. The chloroform is finally dried using anhydrous sodium sulfate.

Examination of the reaction mixture at this point by NMR spectroscopy, reveals that the conversion into amide is approximately 85% complete. Accordingly, the chloroform solution is cooled in an ice-bath and 21 ml. of triethylamine, followed in about minutes by 14.2 ml. of ethyl chloroformate, is added. After a further minutes, 9.8 ml. of 4-methoxybenzylamine is added, and then in another 5 minutes a further 9.8 m1. of 4-methoxybenzylamine is added. The reaction is concentrated in vacuo giving 6- (triphenylmethylamino )-2,2-dimethyl-3-( N-[ 4- methoxybenzyl]carbamoyl)penam, as an amorphous solid.

6-( Triphenylmethylamino )-2,2-dimethyl-3-( chloro- [N-(4-methoxybenzyl)imino]methyl)penam The amide described above is dissolved in 480 ml. of pyridine, and then the solution is cooled to about 5C. To this solution is added dropwise, with stirring during 10 minutes, 108 ml. of thionyl chloride. The reaction mixture is then allowed to warm slowly to ambient temperature for a further 21 hours. All the volatile components are removed in vacuo leaving the crude imino chloride as an amorphous solid. The NMR spectrum (in CHCI of this product shows absorption bands at 4.70 ppm (singlet, C-3 hydrogen), 4.65 ppm (singlet, benzyl hydrogens), 4.30-4.60 ppm (multiplet, C-5 and C-6 hydrogens), 3.75 ppm (singlet, methoxy hydrogens), 1.57 ppm (singlet, C-2 methyl hydrogens) and 1.38 ppm (singlet, C-2 methyl hydrogens).

6-(Triphenylmethylamino )-2,2-dimethyl-3-( l-[4- methoxybenzyl]tetrazol-5-yl)penam The imino chloride described above is re-dissolved in 500 ml. of chloroform and then the solution is cooled to about 5C. in an ice-salt bath. To the solution is then added, with stirring, 160 ml. of trimethylsilyl azide (available from the Aldrich Chemical Company, lnc.). After being allowed to warm to ambient temperature, the reaction mixture is stirred for a further 22 hours. It is then cooled to about 0C. and 2,000 ml. of 1.5N sodium hydroxide solution is added, followed by sufficient additional 1.5N sodium hydroxide to bring the pH of the aqueous to 6.0. The aqueous phase is separated off, and the chloroform phase is washed successively with 5 2,000-ml. portions of water and l 500ml. portion of saturated brine. The chloroform is then dried by filtration through anhydrous sodium sulfate, and finally concentrated to dryness. The residue is triturated with 1,000 ml. of ether, and then filtered off. This affords 150 g. of crude product, m.p. l74-178C. The crude product is purified by re-dissolving it in chloroform and filtering the solution through chromatographic grade silica gel. The chloroform is removed by evaporation in vacuo, and the residue is again triturated with ether. This affords 128 g. of 6-(triphenylmethylamino)-2,2- dimethyl-3-( 1-[4-methoxybenzyl]tetrazol-5-yl)penam as a light tan solid, m.p. 193l 95C. The infrared spectrum (KBr disc) of the product shows an absorption band at 1790 cm (B-lactam carbonyl). The NMR spectrum (in CDCl;,) shows absorption bands at 7.25 ppm (multiplet, aromatic hydrogens), 5.50 ppm (broad singlet, benzyl hydrogens), 5.05 ppm (singlet, C-3 hydrogen), 4.40 ppm (broad singlet, C-5 and C-6 hydrogens), 3.80 ppm (singlet, methoxy hydrogens), 1.45 ppm (singlet, C-2 methyl hydrogens) and 0.70 ppm (singlet, C-2 methyl hydrogens).

In a similar fashion, the following compounds may be prepared:

6-(triphenylmethylamino)-2,2-dimethyl-3-( 1- benzyltetrazol-S-yl)penam,

6-(triphenylmethylamino)-2,2-dimethyl-3-( 1-[2- methoxybenzyl]-tetrazol-5-yl)penam,

6(triphenylmethylamino)-2,2-dimethyl-3-( l-[4- isopropxy-benzyl]tetrazol-5-yl)penam,

6-(triphenylmethylamino)-2,2-dimethyl-3-( l-[3- chlorobenzyl]-tetrazol-5-yl)penam,

6-(triphenylmethylamino)-2,2-dimethyl-2-( l-[3- methylbenzyl -tetrazol-5-yl )penam,

6-(triphenylmethylamino)-2,2-dimethyl-3-( l-[3- chloro-4-methoxybenzyl]tetrazol-5-yl)penam,

6-(triphenylmethylamino)-2,2-dimethyl-3-( l-[ lphenylethyl]tetrazol-5-yl)penam,

6-(triphenylmethylamino)-2,2-dimethyl-3-( 1- furfuryltetrazol-S-yl )penam,

6-( diphenyl-4-fluorophenylmethylamino )-2,2-

dimethyl-3-( l-[4-nitrobenzyl]tetrazol-5-yl )penam,

6-(diphenyl-3-tolylmethylamino )-2,2-dimethyl-3-( l- [4-ethoxy-benzyl]tetrazol-5-yl)penam,

6-(diphenyl-2-methoxyphenylmethylamino )-2,2-

dimethyl-3-( l- 4-phenylbenzyl tetrazol-S- yl)penam,

6-(dipheny1-4-chlorophenylmethylamino)-2,2- dimethyl-3-( ldiphenylmethyl tetrazol-S- yl)penam,

6-(diphenyl-4-bromophenylmethylamino-2,2-

dimethy1-3-( l-[ 2-thienylmethyl ]tetrazol-5- yl)penam,

6-(di[4-methoxyphenyl]phenylmethylamino)-2,2-

dimethyl-3-( l-benzyltetrazol-5-yl)penam,

6-( di[ 3-chlorophenyl ]phenylmethylamino -2,2- dimethyl-3-( l-[4-methoxybenzyl]tetrazol-5- yl)penam,

6-( di[ 2-tolyl ]phenylmethylamino )-2,2-dimethyl-3- l-[ 2,4-dimethoxybenzyl]tetrazol-5-yl )penam,

6-( 4-chlorophenyl] [4- methoxyphenyl]phenylmethylamino-2,2-dimethyl- 3-( 1 4-methoxyphenyl )ethyl ]tetrazol-5 yl)penam,

6-(di[ 4-fluorophenyl] [biphenyly]methylamino )-2,2- dimethyl-3-( 1-[ 1-(4-chlorphenyl )butyl]tetrazo1-5- yl)penam, v

6-(tri[4-tolyl]methylamino )-2,2-dimethyl-3-( l-[(4- methoxyphenyl )phenylmethyl ]tetrazol-5 yl)penam,

6-( tri[ 3-ethoxyphenyl]methylamino)-2 ,Z-dimethyl- 3-( l'[4-ethylbenzyl]tetrazol-5-yl)penam,

6-( diphenyl-[ 3 -bromophenyl methylamino )-2 ,2-

dimethyl-3-( 1-[ 3-furylmethyl]tetrazol-5-yl)penam and -(triphenylmethylamino)-2,2-dimethyl-3-( l-[ 5- methylfurfuryl]tetrazol-5-yl)penam.

EXAMPLE ll 6-Amino-2,2-dimethyl-3-( l-[ 4-methoxybenzyl]tetraZol-5-yl)penam p-toluenesulfonate.

To a stirred slurry of 143 g. of 6- (triphenylmethylamino)-2,2-dimethyl-3-( l-[4-methoxybenzyl]tetrazol --yl)penam in 1,000 ml. of dry acetone is added 45.0 g. of p-toluenesulfonic acid monohydrate, at ambient temperature. The solids slowly dissolve, giving a clear solution. After about 15 minutes, the product starts to precipitate. Stirring is continued for a further 45 minutes after the product starts to appear, and then first crop of product is filtered off and washed with chloroform. The acetone is evaporated to dryness, and the solid residue is slurried for 45 minutes in 300 ml. of chloroform. This affords a second crop of product. The two crops are combined, slurried for 1 hour in 1,000 ml. of chloroform, filtered off, and dried in vacuo giving 123 g. of 6-amino-2,2-dimethyl-3-(1- [4-methoxybenzyljtetrazol-S-yl)penam p-toluenesulfonate, m.p. l74-175.5C. The infrared spectrum (KBr disc) of the product shows an absorption band at 1795 cm. The NMR spectrum (in DMSO-d,,) shows absorption bands at 7.20 ppm multiplet, aromatic hydrogens), 5.80 ppm (multiplet, benzyl hydrogens, C-5 hydrogen and C-3 hydrogens), 5.20 ppm (doublet, C-6 hydrogen), 3.75 ppm (singlet, methoxy hydrogens, 2.35 ppm (singlet, sulfonate methyl hydrogens), 1.70 ppm (singlet, C-2 methyl hydrogens) and 0.85 ppm (singlet, C 2 methyl hydrogens).

In a similar fashion, the amine protecting group may be removed from the compounds of Example 1.

EXAMPLE Ill 6-Amino-2,2-dimethyl-3-(S-tetrazolyl)penam A stirred solution of 32.0 g. of 6-amino-2,2-dimethyl- 3-( 1-[4-methoxybenzyl]tetrazol-S-yl )penam p-toluenesulfonate, and 24 ml. of anisole, in 96 ml. of trifluoroacetic acid is maintained at 40 i 1C. for minutes. The trifluoroacetic acid is then removed rapidly by vacuum distillation. A l20-ml. portion of ether is added to the residue, which produces a white flocculent suspension. The suspension and solvent is cooled to about 0C., and to it is then added, portionwise, 80 ml. of 2N sodium hydroxide, giving two clear phases. The pH of the aqueous phase at this point is about 2.7. The layers are separated, and the ether phase is discarded. The pH of the aqueous phase is raised to 4.1 with 2N sodium hydroxide. This aqueous phase is then washed with 100 ml. of ether and filtered. It is combined with the corresponding aqueous phases from four other identical experiments, and the total aqueous solu tion is lyophilized to give crude 6 -amino-2,2dimethyl- 3-( 5-tetrazolyl)penam. This crude product is slurried in a small amount of water and filtered off. lt is then resuspended in water and brought into solution by raising the pH to 7.4 by the addition of sodium hydroxide solution. The clear solution is extracted with ether and the extracts are discarded. The pH of the aqueous phase is adjusted to 4.1 using dilute hydrochloric acid, and the product which precipitates is filtered off. The infrared spectrum of the product shows an absorption at 1795 cm. lts NMR spectrum (in DMSO-d shows absorption at 5.65 ppm (doublet C-5 hydrogen), 5.20 ppm (singlet, C-3 hydrogen), 4.70 ppm (doublet, C-6 hydro gen), 1.65 ppm (singlet, C-2 methyl hydrogens) and 1.10 ppm (singlet, C-Z methyl hydrogens).

In a similar fashion, the title compound may be prepared from the following compounds:

6amino-2,2-dimethyl-3-( l-[ 2-methoxyben2yl ]tetraZol-S-yUpenam,

14 6-amino-2,2-dimethyl-3-( l-[4-isopropoxybenzyl1tetrazol-5-yl)penam, 6-amino2,2-dimethyl-3-( 1-[3-chloro4- methoxybenzyl ltetrazol-S-yl )penam 6-amino-2,2-dimethyl-3-( 1-[ l-phenylethyl]tetrazol- 5-yl)penam, 6-amino-2,2-dimethyl-3-( l-furfuryltetrazol-S- yl)penam, 6-amino-2,2-dimethyl-3-( 1-[4-ethoxybenzyl]tetrazol-5-yl)penam, 6-amino2,2-dimethyl-3-( l-[4-phenylbenzyl]tetrazol-5'yl)penam, 6-amino-2,2-dimethyl-3-( l-[dipheny1methyl1tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-( l-[ 2-thienylmethyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-( 1-[l-(4- methoxyphenyl )ethyl]-tetrazol-5-yl )penam, 6-amino-2,2-dimethyl-3-( 1-[(4- methoxyphenyl)phenylmethyl]-tetrazol15- yl)penam, 6-amino-2,2-dimethyl-3-( l-[3-furylmethy1]tetrazol- Y 5-yl)penam, 6-amino-2,2-dimethyl-3-( 1-[4-n-hexyloxybenzyl ]tetrazol5-yl)penam, 6-amino-2,2-dimethyl-3-( l-[3,4-

dimethoxybenzyl tetrazol-S-yl )penam, 6-amino-2,2-dimethyl-3-( 1-[5-methyl-2-thienyl]tetrazol-5-yl )penam, 6-amino-2,2-dimethyl-3-( l-[5-methylfurfury1]tetrazol- 5-yl )penam, 6-amino-2,2-dimethyl-3-( 1-[4-biphenylylmethyl]tet razol-5-yl )penam, 6-amino-2,2-dimethyl-3-( l-[ 2,4-

dimethoxybenzyl]tetrazo1-5-yl )penam 6-amino-2,2-dimethyl3-( l-[4-hydroxybenzyl1tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-( I-[Z-hydroxybenzyl ]tetrazol-S-yl )penam, 6-amino-2,2-dimethyl-3-( 1-[ 2.-acetoxybenzyl]tetraz0l-5-yl)penam, 6-amino-2,2-dimethyl-3-( l-[4-acetoxybenzyl]tetrazol-5-yl )penam, 6-amino-2,2-dimethyl-3-( 1-[4- isobutyryloxybenzyl]tetrazo l-5-yl)penam, 6-amino-2,2-dimethyl-3-( l-[4-formyloxybenzyl]tetrazol-5-yl)penam and i 6-amino-2,2-dimethyl-3-( l-[ 2- ethoxymethoxybenzyl tetrazol-5 -yl )penam.

EXAMPLE 1V Preparation of Benzylpenicillin Tetrazole from 6-Aminopenicillin Tetrazole A 2.0 g. sample of -aminopenicillin tetrazole pure by standard hydroxylamine assay) is dissolved in 50 ml. of water by slow addition. of 1N sodium hydroxide. The resulting solution is brought to pH 7, and clarified by filtration. Then 1.22 ml. of phenylacetyl chloride 10% excess) is slowly added with stirring keeping the pH between 6-7 with 0.5 N sodium hydroxide. After stirring for 4 hours the solution is cooled to 10C., adjusted to pH 2 with hydrochloric acid and extracted with chloroform. The combined organic layers ml.) are slowly poured into 300 ml. ofa 6:1 hexane/ether mixture, the resulting white solid filtered,

washed with hexane and dried over P to yield benzylpenicillin tetrazole. The benzylpenicillin tetrazole is used in the following example to provide a source of pure 6aminopenicillin tetrazole.

hydroxide and the mixture incubated at 37C. for periods ranging from 0.5 to 4 hours. The reaction mixture was then analyzed in one the thin layer chromatographic systems described below using appropriate In similar fashion, phenoxymethylpenicillin tetrazole 5 bl k d th ti samples as tandard The ame may be prepared substituting phenoxyacetyl Ch o ide procedure was followed with phenoxymethylpenicillin for phenylacetyl chloride. Also, any of the substituted tetrazole Th f ll i Organisms were f d to tetrazoles may be Similarly acylated except that in hibit deacylase activity with both benzyland phenoxthose Cases where the substituent is subject to hydroly- O ymethylpenicmin tetrazole. sis, a product with an unsubstituted tetrazole ring will I p rettgeri ATCC 9918 be obtamed' P. rettgeri ATCC 31052 EXAMPLE V E. coli ATCC 9637 E. coli ATCC 31030 Screening Microorganisms for Penicillin Deacylase K. citrophilic ATCC 21285 A. cerinus [F0 3268 Culturing Organisms A. aerogenes ATCC 31027 Organisms are maintained on Nutrient Agar slant in tumecens ATCC 6947 2.5 by 15 cm tubes for use. Sterile distilled water (10 subtl hs ATCC 31029 ml.) was added to the surface of the slant containing SpeFleS ATCC 31029 the culture growth and the organisms suspended. Five albdus ATCC 10666 milliliters of the suspension was then introduced into Thin Layer Chromatography one liter of inoculum medium in a 2.8 1. Fernbach flash.

The following systems were employed. They were de- The inoculum medium is prepared by separately auto- Y o veloped with ninhydrin or starch-iodine sprays or a claving solutions A and B at 126 C. for minutes and combination thereof. mixing them.

-' I Systeml Solution A Time 20 minutes 30 Solvent System Water i 950 m], Acetone 90% NZ Amine YTT (crude casein extract) 7.0 g Acetic Acid l0'/( Potassium Hydrogen Phosphate 7.0 g Compound R1 Potassium Dihydrogen Phosphate 3.0 g -aminopenicillanic acid 0.47

Ammonium Sulfate 1.0 g 6-aminopenici11in tetrazole 0.52

BYF 300 (crude yeast extract) 0.55 g benzylpenicillin 0.67

S di m Ci 4 g benzylpenicillin tetrazole 0.70

Magnesium Sulfate Heptahydratc 1.0 g phenoxymethylpenicimn pH phenoxymethylpenigillin terazole 0.70 ystem w Time 1.5 hours Cerelose 7.0 g Solvent System water 50 ml 40 butyl acetate 50.3%

. l-butanol 9.471

' l The broth was then incubated on a rotary shaker at dud 28C. After 24 hours, the inoculum medium (10 ml.) Compound was added to the fermentation broth (2 l.) contained in acid 019 -aminopenicillin tetrazole 0.32 a 4 l. fermenter. The fermentation broth is prepared by benzylpenicillin 0.71 mixing the components below and autoclaving for 60 benzylpenicmi" pheiioxymethylpenicillin 0.68

121C mlnutes at phenoxymethylpenicillin tetrazole 0.72

Fermentation Broth 5O EXAMPLE VI Lactic 8 6-Amino-2,2Dimethyl-3-( 5-Tetrazo1y1)Penam Corn Steel Liquor 20.0 pfimssium Hydrogen Phosphate 95 Produced by Immobilized P. Rettgeri Enzyme. Ammonium Sulfate 2.0 Magnesium Sulfate Hcpwhydmm 02 Benzylper iicillin tetrazole (3.58 g, 10 m. moloes) was PH 6840 suspended in deionized water (400 ml) at 37 C. The pH of the suspension was raised from 3.4 to 8.0 with IN The contents of the fermenter were then incubated 9 hydroxlde' A immPblhzed rettgen penio cillm deacylase (30 g, 200 units/g against benzylpeniin a water bath at 28 C., aerated at a rate of one volume I o 7 lume er minute and Stirred at 1750 RPM th cillin) was added and the mixture maintained at 37 perhvo p and a pH of 8.0 by the periodic addition of 1N sodium a t ree bladed impeller. Cells are generally harvested hydroxide The consumption of Sodium hydroxide by Cenmfugatlon after 24 hours stopped after 2 hours and the mixture was filtered. The Activity Screening immobilized enzyme cake was washed with water. The filtrate and wash were combined and acidified to a pH Intact, whole cells harvested as described above were contacted with a 0.25% by weight solution of benzylpenicillin tetrazole in water. Generally, the cell concentration ranged from about 1 to about 50 g/l. The pH was adjusted to and maintained at 8.0 with 1N sodium of 2.0 with 5N hydrochloric acid. The acidified solution was washed three times with ethyl acetate (150 ml each). The organic layers were combined, dried over magnesium sulfate and evaporated to afford phenylacetic acid (1.09 g, yield) mp 7173C. The nuclear magnetic resonance and infrared spectrum were identical to those of an authentic sample of phenylacetic acid. After extraction, the aqueous base was adjusted to a pH of 4.5 with N sodium hydroxide and concentrated in vacuo at a temperature below 40C. to a final volume of 50 ml at which point a white solid precipitated. The mixture was cooled in an ice bath to 5C. and filtered. The solids were washed with ice water and acetone, and air dried to afford the title compound (1.54 g, 64% yield). The nuclear magnetic resonance and infrared spectra were identical to an authentic sample. A second crop, isolated in a similar manner, yielded an additional 4% of the title compound.

In a similar fashion, immobilized enzymes, isolated from the other microorganisms of interest in the instant invention, are used to deacylate benzyland phenoxymethylpenicillin tetrazoles.

EXAMPLE Vll 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by immobilized P. Rettgeri Enzyme.

Following the methods of Example Vl, benzylpenicillin tetrazole (3.33 g, 9.29 m. moles) was contacted with a wet, immobilized enzyme isolated from P. rettgeri ATCC 31052. After 2 hours, the mixture was filtered and the title compound recovered (1.63 g, 73% yield). Spectrophotometric data were identical to those of an authentic sample.

EXAMPLE Vlll 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by Whole Cells of B. subtilis To 170 ml. of water slurry of whole cells of B. subtz'lis ATCC 31029 was added a 30 ml. suspension of benzylpenicillin tetrazole (4.3 g). The pH was adjusted to and maintained at 8.0 by the addition of 1N sodium hydrox ide and the mixture stirred at 37C. for a period of 3 hours. At this point, thin layer chromatography indicated that deacylation was about 80% complete. The mixture was centrifuged and the supemate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate. The resulting aqueous solution was adjusted to a pH of 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which was filtered and air dried to yield the title compound 1.74 g, 60% yield). The nuclear magnetic resonance spectrum was identical with that of an authentic sample of 6-aminopenicillin tetrazole.

EXAMPLE IX 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by Whole Cells of A. Cerinus EXAMPLE X 6-Amino-2,2-Dimethyl-3-( S-Tetrazolyl )Penam Produced by Whole Cells of Cryptococcus albidus To 100 ml. ofa 2% (w/v) whole cell slurry of C. albidus ATCC 10666 is added 2.15 g. of phenoxymethylpenicillin tetrazole. The pH is adjusted to and maintained at 8.0 by the addition of 1N sodium hydroxide and the mixture is stirred at 37C. for 8 hours. The mixture is then centrifuged and the supemate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate. The resulting aqueous solution is adjusted to pH 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which is filtered and dried in a vacuum.

EXAMPLE XI Enzyme Catalyzed Deacylation of 6-(Z-phenylacetamido)-2,2-dimethyl-3-( l-[4- methoxybenzyl]tetrazol-5-yl)penam Using Proteus rettgeri ATCC 9250 Freeze-dried Cells.

To 4.79 g. (10.0 mmol) of 6(2-phenylacetamido)- 2,2-dimethyl-3-( l-[4-methoxybenzyl]tetrazol-5- yl)penam in 10 ml. of dimethylsulfoxide and ml. of water at pH 8.0 and 37 in a 200 ml., 3-necked, round bottom flask equipped with a magnetic stirrer and a base inlet tube pH electrode from a Radiometer pH stat equipped with an autoburette is added 5.0 g. of freezedried Proteus reltgeri ATCC 9250 cells. The pH of the stirred mixture is adjusted to and maintained at 8.0 by the addition of 0.2 N sodium hydroxide by the pH stat. After 24 hours reaction time, the reaction mixture is extracted with three successive ml. portions of chloroform. The combined chloroform extracts are dried over anhydrous sulfated and evaporated in vacuo to give a yellow solid. This yellow solid is recrystallized two times from chloroform/ethyl acetate solution to give white, crystalline 6-amino-2,2-dimethyl-3-( 1-[4- methoxybenzyl1tetrazol-5 -yl)penam.

The same procedure as described above can be used to deacylate other protected penicillin tetrazoles where, among others, the following groups replace 4- methoxybenzyl:methylyloxymethyl, phthalidyl, methoxycarbonylethyl, phenylsulfonylethyl, ethoxycarbonyl, phenylsulfonyl, and p-chlorophenacyl.

What is claimed is:

1. A process for deacylating a penicillin tetrazole selected from the group consisting of and the salts thereof;

wherein R is the acyl moiety of phenylacetic or phenoxyacetic acid;

R is selected from the group consisting of hydrogen, trialkylsilyl having from one to four carbon atoms in each of said alkyl groups, alkanoyloxymethyl having from three to eight carbon atoms, 1- (alkanoyloxy )ethyl having from four to nine carbon atoms, phthalidyl and a tetrazolylpenam nitrogen protecting group;

and R is selected from the group consisting of hydrogen, trialkylsilyl having from one to four carbon atoms in each of the said alkyl groups, alkanoyloxymethyl having from three to eight carbon atoms, l-(alkanoyloxy)ethyl having from four to nine carbon atoms and phthalidyl;

said process comprising dispersing said penicillin tetrazole in water at a concentration of at least about 0.1% by weight, adjusting the pH of resulting aqueous dispersion to a value between about 5 and 9, contacting the penicillin tetrazole with a deacylase, and maintaining the pH of the solution between about 5 and 9 and the temperature between about 5 and 50C. until the reaction is substantially complete, said deacylase being a deacylase for benzylpenicillin when R is the acyl moiety of phenylacetic acid and said deacylase being a deacylase for phenoxymethylpenicillin when R is the acyl moiety of phenoxyacetic acid.

2. The process of claim 1 wherein said diacylase is introduced into the reaction medium be a member of the group consisting of bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, and enzymes from said bacteria or fungi and said enzymes immobilized on a matrix.

3. The process of claim 1 wherein the concentration of said penicillin tetrazole in said aqueous dispersion is from about 0.1 to by weight.

4. The process of claim 1 wherein the contacting temperature lies between about and C.

5. The process of claim 1 wherein said pH is maintained in the range of about 7.0 to 8.8.

6. The process of claim 1 wherein said deacylase is produced by an organism of the genus Proteus.

7. The process of claim 1 wherein said deacylase is produced by the bacterium Proteus reltgeri.

8. The process of claim 7 wherein the bacterial strain is Proteus reltgeri ATCC 9918.

9. The process of claim 7 wherein the bacterial strain is Proteus rettgeri ATCC 31052.

10. The process of claim 1 wherein said deacylase is produced by an organism of the genus Escherichia.

11. The process of claim 1 wherein said deacylase is produced by the bacterium Escherichia coli.

12. The process of claim 11 wherein the bacterial strain is Escherichia coli ATCC 9637.

13. The process of claim 11 wherein the bacterial strain is Escherichia coli ATCC 31031.

14. The process of claim 1 wherein said deacylase is produced by an organism of the genus Kluyvera.

15. The process of claim 1 wherein said deacylase is produced by the bacterium Kluyvera citriophilia.

16. The process of claim 15 wherein the bacterial strain is Kluyvera citricphilia ATCC 21285.

17. The process of claim 1 wherein said deacylase is produced by an organism of the genus Acetobacter.

18. The process of claim 1 wherein said deacylase is produced by the bacterium Acetobacter cerinus.

19. The process of claim 18 wherein the bacterial strain is Acetobacter cerinus IFO 3268.

20. The process of claim 1 wherein said deacylase is produced by an organism of the genus Aerobacter.

21. The process of claim 1 wherein said deacylase is produced by the bacterium Aerobacter aerogenes.

22. The process of claim 21 wherein the bacterial strain is Aerobacter aerogenes ATCC 31027.

23. The process of claim 1 wherein said deacylase is produced by an organism of the genus Arthrobacter.

24. The process of claim 1 wherein said deacylase is produced by the bacterium Arlhrobacter tumescens.

25. The process of claim 24 wherein the bacterial strain is Art/zrobacter tumescens ATCC 6947.

26. The process of claim 1 wherein said deacylase is produced by an organism of the genus Bacillus.

27. The process of claim 1 wherein said deacylase is produced by the bacterium Bacillus subrilis.

28. The process of claim 27 wherein the bacterial strain is Bacillus subtilis ATCC 31028.

29. The process of claim 1 wherein said deacylase is produced by the bacterium Bacillus species ATCC 31029.

30. The process of claim 1 wherein said deacylase is produced by an organism of the genus Cryptococcus.

31. The process of claim 1 wherein said deacylating agent is produced by the fungus Cryplocuccus albidus.

32. The process of claim 31 wherein the fungus strain is Cryptococcus albidus ATCC 10666.

33. The process of claim 1 wherein said penicillin 'tetrazole is a mixture of tautomers in which R and R are both hydrogen. 

1. A PROCESS FOR DEACYLATING A PENICILLIN TETRAZOLE SELECTED FROM THE GROUP CONSISTING OF
 2. The process of claim 1 wherein said diacylase is introduced into the reaction medium be a member of the group consisting of bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, and enzymes from said bacteria or fungi and said enzymes immobilized on a matrix.
 3. The process of claim 1 wherein the concentration of said penicillin tetrazole in said aqueous dispersion is from about 0.1 to 20% by weight.
 4. The process of claim 1 wherein the contacting temperature lies between about 25* and 45*C.
 5. The process of claim 1 wherein said pH is maintained in the range of about 7.0 to 8.8.
 6. The process of claim 1 wherein said deacylase is produced by an organism of the genus Proteus.
 7. The process of claim 1 wherein said deacylase is produced by the bacterium Proteus rettgeri.
 8. The process of claim 7 wherein the bacterial strain is Proteus rettgeri ATCC
 9918. 9. The process of claim 7 wherein the bacterial strain is Proteus rettgeri ATCC
 31052. 10. The process of claim 1 wherein said deacylase is produced by an organism of the genus Escherichia.
 11. The process of claim 1 wherein said deacylase is produced by the bacterium Escherichia coli.
 12. The process of claim 11 wherein the bacterial strain is Escherichia coli ATCC
 9637. 13. The process of claim 11 wherein the bacterial strain is Escherichia coli ATCC
 31031. 14. The process of claim 1 wherein said deacylase is produced by an organism of the genus Kluyvera.
 15. The process of claim 1 wherein said deacylase is produced by the bacterium Kluyvera citriophilia.
 16. The process of claim 15 wherein the bacterial strain is Kluyvera citricphilia ATCC
 21285. 17. The process of claim 1 wherein said deacylase is produced by an organism of the genus Acetobacter.
 18. The process of claim 1 wherein said deacylase is produced by the bacterium Acetobacter cerinus.
 19. The process of claim 18 wherein the bacterial strain is Acetobacter cerinus IFO
 3268. 20. The process of claim 1 wherein said deacylase is produced by an organism of the genus Aerobacter.
 21. The process of claim 1 wherein said deacylase is produced by the bacterium Aerobacter aerogenes.
 22. The process of claim 21 wherein the bacterial strain is Aerobacter aerogenes ATCC
 31027. 23. The process of claim 1 wherein said deacylase is produced by an organism of the genus Arthrobacter.
 24. The process of claim 1 wherein said deacylase is produced by the bacterium Arthrobacter tumescens.
 25. The process of claim 24 wherein the bacterial strain is Arthrobacter tumescens ATCC
 6947. 26. The process of claim 1 wherein said deacylase is produced by an organism of the genus Bacillus.
 27. The process of claim 1 wherein said deacylase is produced by the bacterium Bacillus subtilis.
 28. The process of claim 27 wherein the bacterial strain is Bacillus subtilis ATCC
 31028. 29. The process of claim 1 wherein said deacylase is produced by the bacterium Bacillus species ATCC
 31029. 30. The process of claim 1 wherein said deacylase is produced by an organism of the genus Cryptococcus.
 31. The process of claim 1 wherein said deacylating agent is produced by the fungus Cryptococcus albidus.
 32. The process of claim 31 wherein the fungus strain is Cryptococcus albidus ATCC
 10666. 33. The process of claim 1 wherein said penicillin tetrazole is a mixture of tautomers in which R2 and R3 are both hydrogen. 